Conclusion

Cld is a haem-based enzyme that selectively and effectively detoxifies chlorite with concomitant O-O bond formation. The removal of the by- product chlorite is essential for survival of perchlorate and chlorate respiring bacteria. Cld from different sources has been described as a tetramer in its native state. However, in our crystal we observed a hexameric Cld ring. Based on the size of the solvent-accessible surface area buried after interaction between the six monomers, the hexamer was classified as a possible quaternary biological assembly. Re-interpreting our chromatographic data we found that based on the experimentally determined elution position, Cld could also be a pentameric or hexameric ring. Native mass spectrometry using a multimeric sample with high enzymatic activity demonstrated the presence of a molecular weight corresponding to a Cld pentamer. Taken all these data together, we conclude that the pentamer is an active state of Cld in solution. We cannot exclude the hexamer to be another

biologically relevant quaternary state although it was impossible to measure its activity due to the presence of the thiocyanate inhibitor. However, since cooperativity between the Cld monomers has been excluded based on kinetics results, indicating the monomers independently catalyse chlorite reduction, the precise quaternary state of the enzyme may be less relevant for enzymatic activity. In this case, different quaternary states might be biologically relevant and enzymatically active. For example, there may be an equilibrium between two states, with a transition influenced by protein concentration, pH or a small molecule or an ion 52;53.

The crystal structure of the inhibited, haem incorporated Cld presented here, gives the first insights into the active site of the O-O bond formation and points at some key residues. A strictly conserved histidine (His170) is the axial haem ligand, while a strictly conserved arginine residue (Arg183) occupies a similar position in the distal part of the active site as Arg48 in cytochrome c peroxidase. Arg183 most likely has an essential role in substrate positioning and activation, as the homologous arginine residue in cytochrome c peroxidase. Trp155, another strictly conserved residue in Cld has been identified on geometrical grounds as the electron donor for the reduction of compound I to compound II. Trp155 is also part of the hydrogen bond network that binds the haem cofactor.

The identification of an anion binding site near the haem is an important finding for the interpretation of the catalytic mechanism. The assumed mechanism involving a compound I intermediate would also generate a ClOǦ anion which must remain close to the active site to perform its nucleophilic attack on the oxy-iron species.

On the basis of the EPR monitored redox titration, the reduction potential for the recombinant Cld haem appeared 135 mV lower than for the wild- type enzyme. However, this does not result in a higher specific activity, as could be expected from the stabilization of the ferric state.

To conclude, our data support the proposed catalytic mechanism for O-O bond formation via high valent oxy-iron species and ClOǦ. Furthermore, we have analyzed the active site of Cld and indicated some key residues for chlorite conversion. Our work demonstrates that a unique biological function, chlorite detoxification, has led to a unique haem enzyme.

A combination of site directed mutagenesis with spectroscopical and structural approaches is now necessary to complement the current catalytic mechanism. Pre-steady state kinetics experiments should be performed to study the formation of the Cld-ClO2Ǧ intermediates.

Coordinates

Coordinates and structure factors have been deposited in the Protein Data Bank (accession code 2vxh).

Acknowledgements

We thank Dr Ir Bert Jansen for data collection and Dr R.A.G. de Graaff for helpful discussions. We are grateful to Dr David Flot and other members of the EMBL-ESRF Joint Structural Biology Group for providing crystallographic data collection facilities and help therewith.

Supplementary data

Figure S1. Determination of the quaternary state. Calibration curves created with size exclusion chromatography on a Superdex column. Data for molecular weights standards elution (open circles) have been fitted to the equations shown.

a) The chromatographically determined Kav for Cld (closed circle) together with the calibration equation y = -0.3665x + 2.18 suggests a molecular weight of 105 kD which if one assumes a globular shape, would correlate with a tetrameric state of Cld in solution. b) The apparent molecular radius of 43 Å as calculated from the equation y = 0.0098x + 0.2596 agrees very well with the value of 44 Å (closed circle) estimated from the crystal structure of the hexameric Cld ring (right inset) using Crysol software54. For completeness, a pentameric Cld constructed by superposing Cld subunits onto the putative Cld pentamer 1t0t is also shown (closed triangle; left inset). The ring structures of Cld therefore elute at a Ve that would also correspond to a globular tetramer in solution.

Figure S1. (legend on previous page)

Figure S2. Stereo representation of the anion binding site. Portion of the final 2.1 Å (2Fo − Fc) electron density map centered on the anion binding site, which is occupied by

HCO3 −

Figure S3. EPR spectra of the samples of the redox titration of Ao Cld. EPR conditions: microwave frequency, 9.388 GHz; microwave power, 126 mW; modulation amplitude, 2.0 mT; temperature, 17.5 K.

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